Bottom Line:
Genetic mapping studies may provide association between sequence variants and disease susceptibility that can, with further experimental and computational analysis, lead to discovery of causal mechanisms and effective intervention.We demonstrated that macrophage depletion in strains with the resistant haplotype led to neutrophil influx and greater weight loss despite a lower pathogen burden.Our results show that genetic mapping and network modeling can be combined to identify causal pathways underlying chlamydial disease susceptibility.

Affiliation: Department of Microbiology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America. miyairi-i@ncchd.go.jp

ABSTRACTGenetic mapping studies may provide association between sequence variants and disease susceptibility that can, with further experimental and computational analysis, lead to discovery of causal mechanisms and effective intervention. We have previously demonstrated that polymorphisms in immunity-related GTPases (IRG) confer a significant difference in susceptibility to Chlamydia psittaci infection in BXD recombinant mice. Here we combine genetic mapping and network modeling to identify causal pathways underlying this association. We infected a large panel of BXD strains with C. psittaci and assessed host genotype, IRG protein polymorphisms, pathogen load, expression of 32 cytokines, inflammatory cell populations, and weight change. Proinflammatory cytokines correlated with each other and were controlled by a novel genetic locus on chromosome 1, but did not affect disease status, as quantified by weight change 6 days after infection In contrast, weight change correlated strongly with levels of inflammatory cell populations and pathogen load that were controlled by an IRG encoding genetic locus (Ctrq3) on chromosome 11. These data provided content to generate a predictive model of infection using a Bayesian framework incorporating genotypes, immune system parameters, and weight change as a measure of disease severity. Two predictions derived from the model were tested and confirmed in a second round of experiments. First, strains with the susceptible IRG haplotype lost weight as a function of pathogen load whereas strains with the resistant haplotype were almost completely unaffected over a very wide range of pathogen load. Second, we predicted that macrophage activation by Ctrq3 would be central in conferring pathogen tolerance. We demonstrated that macrophage depletion in strains with the resistant haplotype led to neutrophil influx and greater weight loss despite a lower pathogen burden. Our results show that genetic mapping and network modeling can be combined to identify causal pathways underlying chlamydial disease susceptibility.

pone-0033781-g001: Association of the Ctrq3 locus with immune parameters and disease status.QTL mapping results for (A) day 6 weight, (B) neutrophils, (C) macrophage activation status, (D) C. psittaci load, and (E) G-CSF on chromosome 11. Ctrq3 is located near 58 Mb on chr 11. Significant (genome-wide adjusted p<0.05) and suggestive (adjusted p<0.63) QTLs are indicated by the solid red and grey lines, respectively. Blue lines indicate the likelihood-ratio statistic (LRS) that the phenotype is associated with the genomic locus. The colored lines following the trend of the LRS show the additive effect of the influence of the locus, with red lines indicating that D alleles increase trait values, while green alleles indicate that B alleles increase trait values.

Mentions:
We infected the C57BL/6J parental strain and 40 BXD strains intraperitoneally, and measured peak C. psittaci load, levels of macrophages and neutrophils in the peritoneal cavity, 32 cytokines on days 3 and 6; and disease status as quantified by the weight change from the day of infection. Strains exhibited a spectrum of disease ranging from 30% weight loss to 10% weight gain over 6 days. Significant variation in cytokine protein expression was detected for 17 of 32 cytokines (all results will be deposited and will be accessible in GeneNetwork, www.genenetwork.org). We confirmed that the previously mapped and cloned Ctrq3 locus on chromosome 11 is a major controller of weight change, macrophage activation status (MAS), level of neutrophil recruitment, and C. psittaci load on day 6 (Figure 1). A novel secondary locus was mapped to distal Chr 1 at ∼190 Mb. This locus modulates levels of several key cytokines—GM-CSF, IL1a, MIP1a, MIP1b, MIP2—but has no effect on disease severity as measured by weight changes (Figure 2). To further investigate the influence of the genetic polymorphisms at Ctrq3, we analyzed the expression pattern of the IRGM2 protein in the peritoneal lavage specimens from infected BXD strains and found that it had two distinct band sizes that are directly correlated with the Ctrq3 genotype [7].

pone-0033781-g001: Association of the Ctrq3 locus with immune parameters and disease status.QTL mapping results for (A) day 6 weight, (B) neutrophils, (C) macrophage activation status, (D) C. psittaci load, and (E) G-CSF on chromosome 11. Ctrq3 is located near 58 Mb on chr 11. Significant (genome-wide adjusted p<0.05) and suggestive (adjusted p<0.63) QTLs are indicated by the solid red and grey lines, respectively. Blue lines indicate the likelihood-ratio statistic (LRS) that the phenotype is associated with the genomic locus. The colored lines following the trend of the LRS show the additive effect of the influence of the locus, with red lines indicating that D alleles increase trait values, while green alleles indicate that B alleles increase trait values.

Mentions:
We infected the C57BL/6J parental strain and 40 BXD strains intraperitoneally, and measured peak C. psittaci load, levels of macrophages and neutrophils in the peritoneal cavity, 32 cytokines on days 3 and 6; and disease status as quantified by the weight change from the day of infection. Strains exhibited a spectrum of disease ranging from 30% weight loss to 10% weight gain over 6 days. Significant variation in cytokine protein expression was detected for 17 of 32 cytokines (all results will be deposited and will be accessible in GeneNetwork, www.genenetwork.org). We confirmed that the previously mapped and cloned Ctrq3 locus on chromosome 11 is a major controller of weight change, macrophage activation status (MAS), level of neutrophil recruitment, and C. psittaci load on day 6 (Figure 1). A novel secondary locus was mapped to distal Chr 1 at ∼190 Mb. This locus modulates levels of several key cytokines—GM-CSF, IL1a, MIP1a, MIP1b, MIP2—but has no effect on disease severity as measured by weight changes (Figure 2). To further investigate the influence of the genetic polymorphisms at Ctrq3, we analyzed the expression pattern of the IRGM2 protein in the peritoneal lavage specimens from infected BXD strains and found that it had two distinct band sizes that are directly correlated with the Ctrq3 genotype [7].

Bottom Line:
Genetic mapping studies may provide association between sequence variants and disease susceptibility that can, with further experimental and computational analysis, lead to discovery of causal mechanisms and effective intervention.We demonstrated that macrophage depletion in strains with the resistant haplotype led to neutrophil influx and greater weight loss despite a lower pathogen burden.Our results show that genetic mapping and network modeling can be combined to identify causal pathways underlying chlamydial disease susceptibility.

Affiliation:
Department of Microbiology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America. miyairi-i@ncchd.go.jp

ABSTRACTGenetic mapping studies may provide association between sequence variants and disease susceptibility that can, with further experimental and computational analysis, lead to discovery of causal mechanisms and effective intervention. We have previously demonstrated that polymorphisms in immunity-related GTPases (IRG) confer a significant difference in susceptibility to Chlamydia psittaci infection in BXD recombinant mice. Here we combine genetic mapping and network modeling to identify causal pathways underlying this association. We infected a large panel of BXD strains with C. psittaci and assessed host genotype, IRG protein polymorphisms, pathogen load, expression of 32 cytokines, inflammatory cell populations, and weight change. Proinflammatory cytokines correlated with each other and were controlled by a novel genetic locus on chromosome 1, but did not affect disease status, as quantified by weight change 6 days after infection In contrast, weight change correlated strongly with levels of inflammatory cell populations and pathogen load that were controlled by an IRG encoding genetic locus (Ctrq3) on chromosome 11. These data provided content to generate a predictive model of infection using a Bayesian framework incorporating genotypes, immune system parameters, and weight change as a measure of disease severity. Two predictions derived from the model were tested and confirmed in a second round of experiments. First, strains with the susceptible IRG haplotype lost weight as a function of pathogen load whereas strains with the resistant haplotype were almost completely unaffected over a very wide range of pathogen load. Second, we predicted that macrophage activation by Ctrq3 would be central in conferring pathogen tolerance. We demonstrated that macrophage depletion in strains with the resistant haplotype led to neutrophil influx and greater weight loss despite a lower pathogen burden. Our results show that genetic mapping and network modeling can be combined to identify causal pathways underlying chlamydial disease susceptibility.